the dynein motor protein complex: structure and dynamics hhmi summer research 2009 with dr. elisar...
TRANSCRIPT
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The Dynein Motor Protein Complex: Structure and Dynamics
HHMI Summer Research 2009
with Dr. Elisar Barbar
By: Jonathan Yih
http://genome.gsc.riken.go.jp/hgmis/graphics/slides/images/Dynein_web.jpg
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GoalImprove understanding of dynein’s structure
http://www.physics.berkeley.edu/research/yildiz/DyneinNew.jpg
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Can lead to greater understanding of basic biological processes
http://dms.dartmouth.edu/compton/photos/photos/Mitotic_spindle_sm.jpg
Mitosis Motor Neuron Disease
http://www.physorg.com/newman/gfx/news/28-researcherss.jpg
Relevance
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Dynein
Motor protein– Chemical energy (ATP) to
mechanical work
Functions– Transport– Organelle positioning– Cell division
http://cjournal.concordia.ca/journalarchives/2006-07/feb_8/008627.shtml
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Made with 3DS Max 9
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Background
A short domain of intermediate chain, IC3, is shown to be disordered but still able to bind LC8 and Tctex-1.
IC3 = IC84-143 Heavy Heads
Heavy Chains
LC8 Tctex-1
IC3
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Hypothesis
The interaction of light chains with IC3 causes small changes in ordered structure that are not limited to the binding site of IC3.
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Barbar 08 Biochemistry
Hypothesis
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Learning the basics
Protein steps:
– Expression/Growth
– Purification
– Cleaning
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/Fig5_18aCationExchColumn.GIF
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Preparing LC8
LC 8 Purification
1 2 3 4 5 6Lane Description1 FT2 Buffer3 50 mM
Imidazole4 100 mM
Imidazole5 350 mM
Imidazole6 MW markers
Learning the basics
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Make Tctex-1 unlabeled
Tctex-1 Purification
1 2 3 4 5 6
Lane Description1 FT2 Buffer3 50 mM
Imidazole4 100 mM
Imidazole5 350 mM
Imidazole6 MW markers
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Make IC3 labeled
1 2 3 4 5 6
15N IC3 Purification
Lane Description1 MW markers2 FT3 Buffer4 50 mM
Imidazole5 100 mM
Imidazole6 350 mM
Imidazole
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IC3/Tctex-1 complex
Titrate IC3 with unlabeled Tctex-1 and collect Nuclear Magnetic Resonance (NMR) dynamics experiments
+ 2
IC3 Tctex-1
?
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Analyzing dynamics data
http://www.chem.unsw.edu.au/research/groups/field/images/two-D-NMR.gif
1H and 15N
Peak 1
Peak 2
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HSQC - correlation of 1H and 15N
Unphased Phased
1H 1H
15N 15N
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Free IC3 IC3 bound to LC8
HSQC interactions
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Nuclear Overhauser Effect data
-6.009-9.380
N HHow “flexible” the N/H bond is
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Acknowledgements
Special Thanks to:– Dr. Elisar Barbar– The Barbar lab group
– Dr. Kevin Ahern– Howard Hughes Medical Institute
Yujuan Song Afua NyarkoGreg Benison Justin HallJessica Morgan